Technical Refolding of Proteins: Do We Have Freedom to Operate? Maria Eiberle, Alois Jungbauer

Technical refolding of proteins: Do we have freedom to operate? Maria Eiberle, Alois Jungbauer

To cite this version:

Maria Eiberle, Alois Jungbauer. Technical refolding of proteins: Do we have freedom to operate?. Biotechnology Journal, Wiley-VCH Verlag, 2010, 5 (6), pp.547. 10.1002/biot.201000001. hal- 00552343

HAL Id: hal-00552343 https://hal.archives-ouvertes.fr/hal-00552343 Submitted on 6 Jan 2011

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Technical refolding of proteins: Do we have freedom to operate?

For Peer Review

Journal: Biotechnology Journal

Manuscript ID: biot.201000001.R1

Wiley - Manuscript type: Review

Date Submitted by the 30-Mar-2010 Author:

Complete List of Authors: Eiberle, Maria; Boehringer Ingelheim RCV Jungbauer, Alois; University of Natural Resources and Applied Life Sciences, Department of Biotechnology

Primary Keywords: Biochemical Engineering

Secondary Keywords: Bioseparation

Keywords: refolding, patent, continuous

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1 For submission to Biotechnology Journal Ms. No. biot.201000001 2 3 4 5 6 7 8 9 10 11 12 Technical Refolding of Proteins, Do we have Freedom to 13 14 15 Operate? 16 1,3 2* 17 Maria K. Eiberle , Alois Jungbauer 18 1 19 Boehringer Ingelheim RCV & Co GmbH, Vienna, Austria 20 2 Department of Biotechnology,For University Peer of Natural ReviewResources and Applied Life Science, Vienna, Austria 21 22 3 current address Rentschler Biotechnologie GmbH, Laupheim, Germany 23 24 25 26 st 27 1 revision 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 * Corresponding Author: 58 59 Mailing address: Muthgasse 18, 1190 Wien, Austria 60 E-mail address: [email protected] Tel.: +43 136006 6226; Fax: +43 1 3697615

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1 Abstract 2 3 4 Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale 5 production of therapeutic proteins, that do not require post-translational modifications. High 6 7 expression yields and simple recovery steps of inclusion bodies from the host cells are 8 9 attractive features in the industrial scale. However, the value of an inclusion body based 10 11 process is dominated by the solubilization and refolding technologies. Scale-invariant 12 13 technologies, economically and applicable for a wide range of proteins are requested by 14 industry. The main challenge is to convert the denatured protein in its native conformation at 15 16 high yields. Refolding competes with misfolding and aggregation. Thus, yield of native 17 18 monomer depends strongly on the initial protein concentrations in the refolding solution. 19 20 Reasonable yields areFor attained atPeer low concentrations Review ( ≤ 0.1 mg/mL). However, that requires 21 large buffer tanks and time-consuming concentration steps. We attempt to give an answer to 22 23 which extent refolding of proteins is protected by patents. Low-molecular mass additives have 24 25 been developed to improve refolding yields through the stabilization of the protein in the 26 27 solution and shielding hydrophobic patches. Progresses were established in the field of high- 28 pressure renaturation and on-column refolding. Mixing times of the denatured protein in the 29 30 refolding buffer were reduced by newly developed devices and the introduction of specific 31 32 mixers. Concepts of continuous refolding were introduced in order to reduce tank sizes and 33 34 increase yields. A few patents covering refolding of proteins will expire soon or have expired. 35 36 This gives more freedom to operate. 37 38 39 40 41 Keywords: Inclusion bodies, refolding, E. coli , recombinant proteins, on column, additives 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Introduction 4 5 6 Approximately 40 % of all biopharmaceuticals are produced in E. coli cells [1]. E. coli cells 7 grow rapidly to high cell densities on inexpensive substrates and well established 8 9 fermentation strategies are attractive for an economic expression in industrial scales. 10 11 Furthermore, the genetic properties of E. coli are well characterized and the strains are easy to 12 13 handle. This explains why E. coli is an economic and efficient production system, and widely 14 15 used for the expression of recombinant proteins [2,3]. However, recombinant proteins are not 16 always folded in their proper and active conformation during protein biosynthesis. For a broad 17 18 majority of heterologous proteins, secretion in E. coli results in 0.5 to 0.8 g L-1 volumetric 19 20 yield [4]. Higher titersFor can be attained,Peer but usually Review require an extensive engineering of the 21 22 fermentation protocol and expression system [5,6,7]. Thus many products on the market are 23 produced as inclusion bodies in the cytoplasm of E. coli , where high fermentation titers can be 24 25 achieved according to Biopharmaceutical Products in the US and European Markets 6th 26 27 edition. Inclusion bodies contain the target protein as insoluble aggregates, present in a kind 28 29 of paracrystalline form. The proteins exist in non-native conformations but with a certain 30 31 content of secondary structure elements [8]. After solubilization with chaotropic buffers in a 32 reducing environment an elaborative renaturation step is required to refold the protein in its 33 34 native and active conformation [9]. Anyway, this technology is widely used. Beside high 35 36 expression yields there are other benefits, that compensate the disadvantage of an additional 37 38 refolding step: Inclusion bodies have a higher density (~1.3 g/mL) than other cellular 39 components and cell debris and can be easily separated and purified by a combination of cell 40 41 homogenization and centrifugation. During expression the target protein accumulates in the 42 43 inclusion bodies and is mostly resistant to proteolytic attacks of cell proteases. After primary 44 45 isolation of inclusion bodies adhesive impurities such as cell debris and host cell proteins can 46 47 be reduced by several wash and centrifugation steps. Finally a high purity of up to 90 % of the 48 product protein can be achieved in inclusion bodies. This simplifies and reduces subsequent 49 50 purification steps [10,11]. 51 52 The process for the production of recombinant protein from inclusion bodies comprises cell 53 54 cultivation and harvest, disruption, recovery of inclusion bodies, solubilization, refolding and 55 reoxidation of disulphide bonds and further purification steps (Figure 1) [11,12]. If inclusion 56 57 bodies contain high amounts of impurities, a denatured purification step of the solubilized 58 59 protein can be performed prior to refolding. Popular methods are ion exchange, size exclusion 60 or metal ion affinity chromatography [13,14,15].

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1 Inclusion bodies are aggregated and densely packed paracrystalline forms of protein. These 2 3 refractile particles are solubilized in high concentrations of chaotropic agents such as urea or 4 guanidine hydrochloride. Reducing conditions are inevitable, as non-native intra- and 5 6 intermolecular disulfide bonds may have been formed in inclusion bodies during translation. 7 8 Solubilization results in a protein in its denatured form. The subsequent step transfers the 9 10 unfolded and reduced protein into conditions, where the formation of native and bioactive 11 12 structures is favored. Of all process steps, refolding is the most crucial step. It decides on the 13 efficiency of an inclusion body based process [16,17,18]. 14 15 Renaturation is initiated by reducing or removing the chaotropic solvent. The yield of the 16 17 refolding step depends strongly on the renaturation conditions such as pH, redox conditions, 18 19 buffer additives and protein concentrations. These conditions have been empirically optimized 20 for each individual protein.For Peer Review 21 22 Most proteins contain cysteine residues that form disulfide bonds, which are required for the 23 24 formation of a native, bioactive structure. For in vitro refolding it is usually essential to add a 25 26 redox buffer system to support the formation of native disulfide bonds. Supplementing the 27 28 refolding buffer with low molecular weight thiol reagents allows the formation of disulfide 29 bonds, as well as the reshuffling of incorrect formed bonds. Generally a combination of a 30 31 reduced and oxidized component is used, for example cysteine/cystine or reduced/oxidized 32 33 glutathione. Suitable ratios must be found to maximize yields [16,19]. Molar ratios of reduced 34 35 to oxidized agents are recommended between 5:1 and 1:1. These ratios provide a suitable 36 redox potential for the formation and reshuffling of disulfide bonds [20,21]. 37 38 However, the correct folding pathway competes often in disadvantage, with aggregation and 39 40 misfolding of the target protein. These two reactions dominate the efficiency of the in vitro 41 42 refolding step. After dilution of the unfolded and reduced protein in a refolding buffer, 43 transient intermediates (I) are formed (Figure 2). Usually, these intermediates are partially 44 45 folded and hydrophobic patches are not completely buried in the core of the protein. There are 46 47 two proposed reaction pathways for these intermediates. One leads to the native 48 49 conformation, where are intramolecular interactions involved. This reaction follows a kinetics 50 51 of first order. The other pathway directs to aggregates where intermolecular interactions are 52 responsible for a second- or higher-order reaction. This kinetic partitioning explains 53 54 decreasing refolding yields with increasing initial concentrations of unfolded and reduced 55 56 protein [22,23,24,25]. The decline of the refolding yield with rising concentrations of the 57 58 denatured protein is demonstrated in Figure 3 for α-lactalbumin and a single-chain antibody 59 fragment (scFv). The susceptibility of aggregation is here clearly higher for the scFv. At 60 concentrations ≤ 0.1 mg/mL both proteins can be totally renatured. However, increasing the concentration of the denatured protein leads to a tremendous loss of native protein. As

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1 aggregation results from nonspecific interactions between hydrophobic regions or partially 2 3 folded structures of different polypeptide chains, it is usually irreversible [26]. Suppressing 4 aggregation is therefore an inevitable step to achieve high yields of native and active protein. 5 6 A common process for protein refolding in industry is batch dilution at very low protein 7 8 concentrations ( ≤ 0.1 mg/mL) [20,27]. However, this requires large volumes of refolding 9 10 buffer, huge reactors and additional concentration steps. Beside high costs for time- 11 12 consuming steps, waste disposal has also to be taken into account. Therefore the refolding 13 step needs to be carefully optimized, to gain a simple and cost-effective bioprocess [16]. As 14 15 consequence, conditions have to be found, where the hydrophobic interactions are suppressed 16 17 and refolding to the native protein is favored. Unwanted side reactions as misfolding and 18 19 aggregation have to be decelerated. Thus a lot of research and development have been done to 20 optimize various parametersFor inPeer the refolding Review step. One approach is the search and 21 22 development of new additives, supporting the solubility after dilution and shield hydrophobic 23 24 patches during folding. Other approaches focus on the addition mode of the denatured protein 25 26 to the renaturation buffer. Reduced mixing times result in lower local protein concentrations 27 28 and decrease aggregation rates [10]. Refolding under high pressures or on a column matrix is 29 also a possibility to attain high recovery rates in a renaturation process [9,10]. 30 31 It is not the purpose of the manuscript to give advice how to circumvent existing patents. We 32 33 want to draw attention to patent literature, which may prevent the application of certain 34 35 refolding procedures. Mainly we focus on process patents, less on patents expressing a certain 36 protein a unique refolding conditions. 37 38 39 40 Refolding conditions and processes 41 42 43 Refolding by direct dilution 44 45 The simplest method to refold proteins is the addition of the unfolded and reduced protein 46 47 directly to the refolding buffer. In biopharmaceutical manufacturing this is a widely used 48 49 method, as this addition mode is cheap and easy to scale and validate, respectively. Due to the 50 51 aggregation characteristics of the proteins, concentrations are kept low (usually ≤ 0.1 mg/mL) 52 53 to attain reasonable refolding yields. Higher concentrations enhance the probability of 54 collisions of unfolded or partially folded protein and lead to higher aggregation rates. Thus 55 56 working at higher concentrations demand a controlled addition of the denatured protein to the 57 58 renaturation buffer. Aggregation is reduced, if low local protein concentrations are rapidly 59 60 achieved in the refolding tank. Therefore, short mixing times are inevitable for an efficient process. To increase the yield of native protein, dilution can be done as fed-batch and continuous dilution. Several groups have claimed these methods but patent protection have 5 Wiley-VCH Biotechnology Journal Page 6 of 28

1 been recently expired [28,29]. In a stirred tank various parameters influence the refolding 2 3 yield, as the intensity of mixing, the injection rate, the injection point, the concentration of 4 denatured protein and the total protein concentration [30,31]. For example, lysozyme shows 5 6 higher refolding yields in fed-batch addition compared to simple batch dilution [32,33]. 7 8 Another continuous solution was filed by Buus et al. in 2002 [34]. In a mixing chamber the 9 10 denatured protein is mixed with the refolding buffer and the refolded protein is recovered 11 12 through a connected expanded bed absorption column. A benefit is here the possibility to 13 recycle the refolding buffer [35]. As mentioned, the intensity of mixing affects the refolding 14 15 yield. To avoid aggregation in the tank, mixing times must be shorter than the reaction rate of 16 17 aggregation. However, in industrial scales large tank reactors exhibit low mixing efficiencies. 18 19 Efforts to improve mixing in large scale are therefore inevitable [31]. A scaleable, efficient 20 mixing device is an oscillatoryFor flowPeer reactor, inventedReview by Middelberg et al. [36]. The mixing 21 22 chamber is a column, that contains the refolding buffer. It is divided in eight sections by seven 23 24 flat ring baffles. The fluid is oscillated through a piston at the bottom of the column. Mixing 25 26 is controllable through the frequency and the amplitude of the piston. Unfolded protein can be 27 28 fed continuously into the column, either at one or two feed points. Compared to a standard 29 fed-batch mode in a stirred tank, refolding yields could be almost doubled by this approach 30 31 [37]. Another possibility to control the intensity of mixing is to involve a static mixing device 32 33 which was protected by patents of St. John et al. [38] and Schlegl [39]. Here, the refolding 34 35 buffer is pumped through a static mixer. The concentrated denatured protein is added to the 36 conduit via the inlet upstream of the mixing device. This system achieves at least as good 37 38 yields as conventional methods. It has the advantages of scalability, higher throughput and 39 40 robustness of the process. Schlegl extended the approach of a static mixer through the 41 42 recirculation of the renaturation buffer (Figure 4). Mixing times can be adjusted by the feed 43 time of the denatured and reduced protein solution and by the recirculation flow. A fast 44 45 collapse of the protein is assumed immediately after folding is initiated. The resulting 46 47 conformation contains native-like secondary structures and is called the “molten globule” 48 49 intermediate. These intermediates are often stable under non-denaturing conditions [40]. 50 51 Through the use of a plug flow reactor prior to the refolding tank, the residence time can be 52 adjusted, necessary for the formation of a stable intermediate. This stabilized conformation is 53 54 less prone to aggregation while the buffer recirculates and further denatured protein is added. 55 56 Another refolding method is reversed dilution. Most dilution methods feed the denatured 57 58 protein solution to the refolding buffer. In reversed dilution the concentration of the 59 denaturant as well as of the protein is decreased simultaneously, as the refolding buffer is fed 60 to the solubilized inclusion bodies. He et al. used this approach to refold a staphylokinase variant [41].

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1 Current patent literature suggests that fed-batch and simple dilution is not covered by patents, 2 3 but for several mixing devices patents have been filed. 4 5 6 Solubilization of inclusion bodies 7 8 Solubilization and refolding are often interrelated. The solublization agent influences the 9 10 refolding conditions. Earlier studies on the structure of inclusion bodies disclosed native or 11 12 native-like structures coexisting with β-forms [42,43]. If the inclusion bodies can be 13 14 solubilized under conditions where these structures can be conserved, refolding may results in 15 clearly higher yields. Conventional solubilization agents are high concentrations of chaotropes 16 17 like urea or guanidine hydrochloride that denature the target protein completely. Thus gentle 18 19 solubilization methods are necessary to keep the secondary native structures that occur in the 20 For Peer Review 21 inclusion bodies. For example, McCoy patented a solubilization process for somatotropin 22 23 inclusion bodies. These inclusion bodies are soluble in low concentrations of urea (around 2 24 M) at a pH of 12 [44]. Another uncommon solubilization approach was filed by Yong-Jun et 25 26 al. [45]. They used organic solvents such as n-propyl alcohol or isopropyl alcohol including 27 28 0.05 % β-mercaptoethanol at a pH around 12 and temperatures of 30 °C and higher. 29 30 Surfactants as N-lauroyl-sarcosine or sodium dodecyl sulfate (SDS) are also used as 31 32 solubilizers that do not disrupt existing structures. However, the use of surfactants is usually 33 avoided as it requires extensive purification steps for the removal from the protein solution 34 35 [17,46]. A total recovery of the target protein from the inclusion bodies is a fundamental goal 36 37 in the overall process. Using gentle solubilization methods may result in low recovery yields. 38 39 Additionally misfolded conformations and multimers in the inclusion bodies can not be 40 dissolved and precipitate immediately after refolding is initiated [47]. Aggregation of proteins 41 42 can be suppressed if definite pH ranges are chosen for solubilization and refolding. At a pH 43 44 far away from the isoelectric point (pI) a protein is highly charged. Charge repulsion occurs 45 46 and prevents aggregation [48]. Denaturing and refolding at alkaline pH and subsequent slow 47 reduction of the pH near the pI can support renaturation and was invented by Xinli [49]. 48 49 Pizarro et al. filed a patent for a process that also focuses on the pH in the solubilization and 50 51 renaturation process [50]. Inclusion bodies were solubilized at pHs greater than 9 and 52 53 refolding was also accomplished at strong alkaline conditions (pHs between 9 and 11). 54 55 It seems that protein solubilization with chaotropic agents is not covered by patents and we 56 have a freedom to operate. When it comes to solubilization a high pH the filed application of 57 58 Pizarro et al. must be taken into consideration 59 60

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1 Refolding buffer composition: Additives and folding aids 2 3 Direct dilution of the denatured and reduced protein in the refolding buffer is a simple and 4 5 fast method. However, renaturation steps require a carefully optimized buffer composition to 6 7 attain reasonable yields. Appropriate additives play here a decisive role. They have to meet 8 following requirements: (1) Improve clearly the refolding yield, (2) preserve the cost- 9 10 effectiveness of the overall process, (3) do not disturb subsequent purification steps and (4) 11 12 are removable from the final product during the purification process. 13 14 Hydrophobic interactions and hydrogen bonds are responsible for the aggregation of proteins. 15 16 Primarily, unfolded proteins and folding intermediates are prone to aggregation. The addition 17 of low molecular additives or detergents to the refolding buffer reduces aggregation and in 18 19 consequence precipitation during the renaturation process. Usually, these compounds can be 20 For Peer Review 21 easily removed after the refolding step. Likely, they support the solubility and stability of the 22 23 native, denatured and intermediate states [51]. The most frequently used additive is L-arginine 24 and it seems that the addition of this compound for enhancing solubility is not protected by a 25 26 patent. Usually, it is added to the refolding buffer in concentrations of 0.4 to 0.8 M [52,53]. 27 28 Liu et al. [54] reported, that L-arginine temporarily stabilizes the denatured proteins as well as 29 30 early partially folded intermediates and refolded protein. Through specific interactions L- 31 32 arginine slows down conformational movement and consequently protein-protein association 33 during the refolding process [55]. L-arginine as folding additive, especially in industrial scale, 34 35 is very costly. Chaotropic agents as guanidine hydrochloride and urea can also be used as 36 37 refolding additives. This, however, requires denaturant concentrations that do not destabilize 38 39 the native state of the protein [51]. One explanation of the mode of action of urea and 40 guanidine hydrochloride as protein stabilizing agents is the preferential interaction theory. It is 41 42 considered, that protein stabilizing factors are based on direct protein-denaturant interactions 43 44 [56]. Pike and Acharya investigated the interactions between urea and lysozyme [57]. Subtle 45 46 conformational changes took place within the crystal structure of lysozyme upon exposure to 47 urea. These changes were observed at regions of the surface of the molecule, that are known 48 49 to be relatively flexible. Previous studies indicated that urea and guanidine hydrochloride, 50 51 present in non-denaturing concentrations, interact with the protein through multiple hydrogen 52 53 bonds [56,57,58]. 54 55 Shiraki et al. [59] invented a new refolding additive which is a derivative of arginine. They 56 proofed, that L-argininamide is a better refolding additive compared to the widely used L- 57 58 arginine [60]. For lysozyme the refolding yield was 1.7-fold higher in the presence of 59 60 argininamide than for arginine. In the case of bovine carbonic anhydrase the improvement of the final refolding yield was 1.4-fold.

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1 A new additive was invented by Flowers and Summers [61]. Hen egg white lysozyme could 2 3 be successfully refolded at high protein concentrations using ethylammonium nitrate (EAN). 4 Examinations of the effect of EAN on the thermal properties of the enzyme showed, that EAN 5 6 acts as denaturant. Proteins can be denatured with EAN and refolded by simple dilution, 7 8 resulting in reasonable refolding yields at high protein concentrations. It is supposed, that the 9 10 ethyl residue of EAN interacts with the hydrophobic patches and suppresses aggregation. The 11 12 charged ammonium group is assumed to stabilize the secondary structure through electrostatic 13 interactions. EAN has the main advantage of being easily removed by desalting methods [62]. 14 15 A similar approach was patented by Peters et al. [63]. They presented that chemical denatured 16 17 protein can be successfully refolded in presence of secondary and tertiary amines. Adding 18 19 triethanolamine-H2SO 4 in concentrations around 1 M showed a good refolding efficiency for 20 recombinant interleukin-4.For For bovinePeer pancreatic Review trypsin Tris(hydroxymethyl)-aminoethane 21 22 (TRIS) in combination with H 2SO 4 appeared as suitable agent for renaturation. 23 24 Fluorine compounds are also potential renaturants, as invented by Lohr et al. [64]. They could 25 26 renature human serum albumin after heat denaturation by adding trifluoroethanol to the 27 28 solution. Examinations with different fluorine derivatives indicated, that compounds with a 29 trifluoromethyl group and a vicinal hydroxyl group are most effective for refolding. 30 31 Other additives, used in protein folding technology are cyclodextrins. These cyclic glucose 32 33 oligosaccharides are able to prevent aggregation through non-covalent inclusion complexes 34 35 with the hydrophobic patches of aggregation prone intermediates. Patents are filed, using this 36 technology [65,66]. Refolding of bovine carbonic anhydrase and recombinant endostatin 37 38 showed much higher yields, if α-cyclodextrin was added to the renaturation buffer [67]. 39 40 Cyclodextrins are also applied in the approach “artificial chaperoning”. This concept was 41 42 introduced by Rozema et al. [68] in 1995 and is similar to the function of the natural 43 44 molecular chaperones. It comprises two steps: In the first step detergent molecules are added 45 to capture the non-native species of the protein by forming a protein-detergent complex. 46 47 Aggregation and also native refolding is disabled. To start renaturation ß-cyclodextrin is 48 49 added in the second step. It strippes off the detergent from the protein-detergent complexes 50 51 and allows the protein to form its native conformation. There are three major cyclodextrins 52 53 with six, seven and eight glucose units ( α-, β-, γ-cyclodextrin, respectively). Cycloamylose 54 has more glucose units, usually between 17 and hundred. Machida et al. [69,70] invented the 55 56 use of cycloamylose as additive in refolding experiments. In combination with dedicated 57 58 detergents superior refolding yields could be achieved in comparison to the corresponding 59 60 conventional α-, β- and γ-cyclodextrins. Another stripping agent was reported by Khodagholi et al. [71]. They used alginate instead of cyclodextrins. Alginate is a linear polysaccharide that consists of β-D-mannuronic acid and α- 9 Wiley-VCH Biotechnology Journal Page 10 of 28

1 L-guluronic acid. Electrostatic forces provoke the interaction of the alginate with oppositely 2 3 charged detergents [72]. Refolding of alkaline phosphatase in dodecyl trimethlyammonium 4 bromide (DTAB) and subsequent stripping of the charged detergent with alginate resulted in 5 6 high recovery yields of native protein [71]. 7 8 Reversed micelles are also an option for the renaturation of protein derived from inclusion 9 10 bodies. They are formed, when mixtures of surfactants, water and nonpolar solvents are 11 12 combined in defined concentrations. The surfactant aerosol OT (AOT, sodium dioctyl 13 sulfosuccinate) is very common for this system. Aqueous nanoscaled droplets are formed, 14 15 stabilized by surfactants. Through micelles, protein molecules are separated of each other. 16 17 Intermolecular interactions are reduced and aggregation is prevented. The polar groups are 18 19 concentrated inside, whereas the lipophilic groups extend to the non-polar organic solvent. 20 The size of the aqueousFor droplet insidePeer of the micelle Review depends on the conditions of the system. 21 22 Variables as surfactant to water ratio, ionic strength, surfactant and protein concentration have 23 24 to be varied until the size of a micelle conforms to the size of a protein molecule. The total 25 26 process comprises four steps: (1) The denatured protein has to be transferred into the micelles, 27 28 (2) the denaturant has to be removed, (3) folding agents as redox reagents can be added and 29 (4) extraction of the renatured protein from the micelles. RNase A was successfully refolded 30 31 with this system [73,74]. Compared to the conventional dilution method Sakono et al. [73] 32 33 could improve refolding yields from 40 to 100 %. But the refolding kinetics was slower with 34 35 the reversed micelle mediated system. To overcome this problem, Sakono et al. added the 36 molecular chaperone GroEL into the reversed micelles. The kinetic was significantly 37 38 improved. After one hour more than 80% activity was achieved, whereas without GroEL only 39 40 20 % could be recovered. 41 42 Recently, new refolding additives were published by Pitner et al. [75]. They used ionic liquids 43 with a specific distribution of the electron density for protein refolding. Ionic liquids or liquid 44 45 salts consist usually of an organic cation and an inorganic anion. The cation has at least one 46 47 electron donor region and one positive charged electrostatic region. Pitner et al. refolded 48 49 several proteins in presence of ionic liquids. They showed the potency of ionic liquids in 50 51 comparison to conventional additives, as for example L-arginine. 52 Mostly the exact mechanism of action of refolding additives is a supposition or even unclear. 53 54 This makes it inevitable to determine suitable additives as well as refolding conditions for a 55 56 certain protein by trial and error. It is also not clear, how far the protection of additives 57 58 reaches. The current practice of patent offices is to strictly limit the invention to the presented 59 example. Thus it is difficult to demonstrate, that a certain additive is suited for all proteins. In 60 particular certain compounds are protected by patents although a general patent on the addition of additives does not exist. An overview of additives is provided in Table 1. We

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1 assume that there are many more compounds have been claimed as additives but often only 2 3 connection with a certain protein. 4 5 6 High-pressure disaggregation and folding 7 8 Unfolding of many proteins under high hydrostatic pressure has been known for more than 90 9 10 years [76]. Pressure is a useful tool to study thermodynamics of proteins, as well as unfolding 11 12 and refolding processes [77,78]. In general, dissociation of multimeric proteins is facilitated at 13 14 pressures between 1000 and 3000 bar. For total unfolding pressures up to 8000 bar are usually 15 necessary. The specific volume of a protein state, formed at high pressures, dominate the 16 17 renaturation process. Simple thermodynamics is responsible for this observation. The pressure 18 19 dependent change of Gibbs free energy between two states corresponds to their difference in 20 For Peer Review 21 specific volume. Several factors are assumed to cause these volume decreases. Inside folded 22 proteins or at interfaces of oligomeric proteins there are cavities and internal voids, that favor 23 24 unfolding or dissociation. These intra- and intermolecular cavities are dissected during 25 26 unfolding processes. Electrostatic interactions are disrupted and the electrostriction of water 27 28 molecules around free charged groups is caused. Moreover, hydrophobic patches and polar 29 30 groups are exposed to hydration [79]. High pressure destabilizes native proteins. In 31 magnitude, the occurring change of the specific volume during unfolding is quite small. It is 32 33 only 0.5 to 1 % of the total specific volume, but nevertheless significant [80]. 34 35 Pressure induced unfolding is favored as it is reversible and usually only low concentrations 36 37 or even no chaotropic agents have to be added. Conventional solubilization techniques of 38 inclusion bodies comprise highly concentrated guanidine hydrochloride or urea buffers. 39 40 Usually proteins are completely unfolded in these chaotropic agents. The combination of high 41 42 hydrostatic pressure and low concentrations of guanidine hydrochloride prevents aggregation 43 44 during refolding [81]. Applying high pressure to aggregates or inclusion bodies results in 45 solubilization under gentle conditions. Folding can be started from structures, that are less 46 47 interrupted or even possess some secondary structure. Studies with RNase A showed, that 48 49 pressure denatured RNase A keeps a significant degree of secondary structure. Generally it 50 51 can be stated, that the pressure denatured states of proteins contain more secondary structure 52 53 than the temperature or chemical denatured states [77]. 54 β-lactamase was successfully refolded from inclusion bodies applying 2000 bar for 48 h, as 55 56 shown an example of a patent, filed by Randolph et al. [82,83]. Catalytic activity was 57 58 observed even without a chaotropic agent in the renaturation buffer. At higher concentrations 59 60 of guanidine the total protein concentration increased and the soluble amount of β-lactamase as well as the recovered activity remained nearly constant. Applying high pressures in the absence of guanidine hydrochloride can be also used as purification step. The total solubilized 11 Wiley-VCH Biotechnology Journal Page 12 of 28

1 protein increased with the concentration of guanidine, whereas the solubilized concentration 2 3 of the target protein β-lactamase remained the same. As renaturation tool high-pressure has 4 5 the advantage, that it dissociates aggregates while it favors the native conformation. During 6 refolding it prevents and reverses aggregation, which has already taken place. This technology 7 8 was invented by Robinson et al. [84]. Additives as urea of guanidine hydrochloride can 9 10 support this process. That is a major benefit compared to conventional refolding methods, as 11 12 dilution or dialysis. Aggregation is reversible and higher yields of native protein can be 13 achieved. Even high protein concentrations lead to reasonable refolding yields. As for 14 15 conventional refolding methods, like dilution, refolding conditions have to be optimized for 16 17 high pressure renaturation as well. Additives and redox systems have to be tested to improve 18 19 refolding yields. Using glycerol as folding additive for the enzyme rhodanese in combination 20 For Peer Review 21 with the high-pressure technology nearly doubled the renaturation yield [85]. Other 22 parameters as temperature or agitation during pressure treatment can also promote refolding 23 24 yields, as patented by Randolph et al. [86]. They showed, that recombinant human growth 25 26 hormone (rhGH) totally refolded at 60 °C and 2000 bar. Stirring the solution at these 27 28 conditions was found to increase the refolding yield as well as the folding kinetics. Refolding 29 at high pressures in the presence of specific binding agents was filed by Seefeldt et al. [87]. 30 31 As binding agents small organic molecules, polypeptides and nucleic acids are a possibility 32 33 for improvements of renaturation yields. 34 35 In the case of biopharmaceutical preparations, aggregates can induce immune responses, 36 37 including anaphylactic reactions in patients. The consequences can be fatal. Aggregates do 38 not only occur during refolding. In processing steps as filtration, ultrafiltration, 39 40 chromatography, vial filling, crystallization and so on, protein aggregation can be observed 41 42 [88]. 43 44 The use of high-pressure could be a valuable tool in the preparation and storage of 45 pharmaceutical drugs to prevent aggregation. In addition, high-pressure technology to 46 47 disaggregate and refold proteins is efficient and cost-effective. Refolding at high protein 48 49 concentrations does not require large-scale dilution and additional concentration steps. The 50 51 plant throughput can be increased which is favorable in industrial production processes. 52 Currently several patents have been filed to protect high pressure refolding of recombinant 53 54 proteins and dissolution of aggregates. In this respect we assume a very limited freedom of 55 56 operation and a contact with the owners is advisable. 57 58 59 On-column refolding 60 There are three different approaches for on-column refolding: (1) Dilution of denaturants by size exclusion chromatography, (2) retention of the denatured protein on a chromatographic 12 Wiley-VCH Page 13 of 28 Biotechnology Journal

1 phase and subsequent removal of the denaturant and (3) immobilization of a folding catalyst 2 3 on a chromatographic phase, where the column acts as a catalyzed refolding reactor [89]. On- 4 column refolding offers the advantage of refolding and simultaneous purification. 5 6 Refolding using size exclusion chromatography is based on a gradual removal of the 7 8 denaturant. Aggregates, intermediates and native protein are separated by their different 9 10 diffusion properties in the stationary phase. The denatured protein has a high hydrodynamic 11 12 radius and is excluded from the gel pores. During refolding the protein size decreases and gel 13 pores are easier accessible. Gu et al. presented, that the overall refolding yields of a single- 14 15 chain antibody fragment (scFv) with SEC are comparable to yields achieved with batch 16 17 dilution [90]. Using an urea gradient resulted in slightly higher yields. The highest yield was 18 19 obtained with a combined urea and pH gradient. However, refolding by SEC has the key 20 advantage over conventionalFor dilution, Peer that the materialReview on the column is fractionated by size. 21 22 A new method applying SEC for refolding is a continuous process. Intermediates and 23 24 aggregates are separated from the native refolded protein and can be reconstituted to the feed 25 26 solution. An extensive study was done with α-lactalbumin as model protein using pressurized 27 28 continuous annular chromatography (Figure 5). Through recycling of intermediates and 29 aggregates the refolding yield could be improved considerably, which was shown and 30 31 patented by Necina et al. [91,92]. 32 33 Adsorption of a denatured protein on a solid matrix is supposed to prevent aggregation 34 35 through separation of the individual protein molecules from each other during refolding. This 36 approach was firstly invented by Creighton [93]. After adsorption of the unfolded protein on 37 38 an ion exchange matrix he initiated refolding. As for SEC, P-CAC with an ion-exchange resin 39 40 achieved better refolding yields for α-lactalbumin, especially when aggregates were 41 42 reconstituted to the refolding step [79,94]. Another method is dual gradient ion-exchange 43 chromatography (IEC). The denatured protein is eluted by gradual decrease of the denaturant 44 45 concentration and increase of the pH. The protein refolds gradually and compared to batch 46 47 dilution higher refolding yields can be attained [95]. Refolding conditions using ion-exchange 48 49 have to be carefully optimized. Otherwise non-specific protein-matrix interactions can occur 50 51 and no refolding takes place. However, if optimal conditions are found, denatured protein can 52 be refolded at significantly higher concentrations as in batch dilution [84]. The principle of 53 54 protein adsorption on an ion exchange matrix can be used in expanded bed adsorption (EBA) 55 56 chromatography. In EBA chromatography the void fraction is increased which allows the 57 58 application of particle containing feedstocks. Especially in large scale applications EBA 59 chromatography is a useful tool, has large volumes can be processed. Compared to 60 conventional on-column refolding with an ion exchange resin, similar refolding yields can be attained with EBA chromatography [96].

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1 Simulated moving bed has been used for continuous refolding of proteins [97, 98] but 2 3 it seems that the method has been never claimed for on-column refolding. Still one might 4 consider that certain configurations or column designs for SMB may be protected by patents. 5 6 Introducing a N- or C-terminal polyhistidine-tag allows refolding on a solid matrix 7 8 based on immobilized metal affinity chromatography (IMAC). Immobilized divalent metal 9 2+ 2+ 10 ions (for example Ni or Cu ) form high-affinity complexes with the polyhistidine-tag in 11 12 presence of high concentrations of denaturants as guanidine or urea. IMAC offers the 13 advantage, that solubilized inclusion bodies are firstly purified and then refolded. Applying 14 15 IMAC offered superior results for the purification of an anti-TNF α-scFv. A refolding yield of 16 17 77 % and purity of 95 % could be obtained [99]. The potency of this method was proofed for 18 19 several proteins, containing a polyhistidine-tag [100,101,102]. 20 Hydrophobic interactionFor chromatography Peer (HIC) Review is also an option for on-column refolding. 21 22 Aggregation can be suppressed through hydrophobic interactions between ligands of the 23 24 matrix and protein molecules. For lysozyme refolding yields increased with rising 25 26 hydrophobic strength of the resin. Adding glycerol to the eluent improved the specific activity 27 28 of the renatured lysozyme [103]. 29 Another set-up for on-column refolding is, to immobilize folding catalysts and 30 31 artificial chaperones. In vivo conditions are mimicked and supposed to improve refolding 32 33 yields. Tsumoto et al. [104] used the foldase oxidoreductase, immobilized on N- 34 35 hydroxysuccinimide-activated Sepharose 4FF, to refold a single-chain antibody fragment and 36 prevented the aggregation of the target protein in the refolding step. An engineered chaperone 37 38 has been also used for improvement of refolding [108,109]. This strategy is not well accepted 39 40 in industry since it is expensive and a lot of chaperone must be produced because they act in a 41 42 stoichiometric manner. Patents have been withdrawn and in this respect we also have freedom 43 to operate, presumably somebody finds an inexpensive alternative to produce mini- 44 45 chaperones. 46 47 A novel approach using zeolite was invented by Mizukami et al. [105,106]. Zeolites 48 49 are crystalline porous aluminosilica compounds. These tectosilicates are made up of AlO 4 and 50 51 SiO 4 tetrahedra that share the corners. In industry, zeolites are widely used as cationic ion 52 exchangers and catalysts. The main advantage of zeolite is, that proteins can be adsorbed in 53 54 the presence of denaturants. The zeolite is suspended in the denatured protein solution and 55 56 after washing away the chaotropic agent, the target protein can be desorbed with buffer 57 58 containing detergents as polyethylene glycol (PEG) and Tween 20. 59 On-column refolding provides a good alternative to common methods [107] especially 60 when the refolding yield in batch refolding is very low. The Creighton patent [92] covering a wide range on column refolding methods has a priority date of April 1 st 1986 and is

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1 meanwhile expired. So we assume freedom of operation for a conventional on column 2 3 refolding passing a denatured protein solution over a chromatography column and elution by a 4 kosmotropic buffer. Tough several more sophisticated including continuous chromatographic 5 6 methods have been filed (Table 1) a general continuous chromatography refolding procedure 7 8 has not been patented. In particular continuous annular chromatography [92] for continuous 9 10 refolding has been claimed. 11 12 13 14 Conclusion 15 16 E. coli is a widely used expression system for the heterologous proteins. The detailed 17 18 knowledge of the genetics, simple cultivation conditions and short generation times are 19 20 attractive in commercialFor and research Peer applications. Review However, overexpression leads mostly to 21 22 the accumulation of insoluble inclusion bodies in the cytoplasm of E. coli and recovery of 23 native protein requires an elaborative renaturation step. Thus, it is desirable to establish 24 25 refolding methods, that are applicable for a wide range of different proteins. New approaches 26 27 and methods have to be investigated to explore their potential as “generic” refolding tools. For 28 29 manufacturing scale novel concepts have to be evaluated regarding their scalability and 30 31 economic features. In general, batch and fed batch refolding methods are not covered by 32 patents. The use of improved solubilization and refolding additives is broadly covered, and it 33 34 is advisable, to check patent literature to avoid infringement. A similar situation has been 35 36 found for continuous refolding. The freedom to operate is given for technical refolding. There 37 38 is enough room, to exploit the power of inclusion body technology for further innovative 39 protein products. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 Fermentation and cell 3 4 harvest 5 6 7 Cell disruption and 8 9 IB isolation 10 11 12 13 IB washing and 14 centrifugation 15 16 17 18 19 IB solubilization 20 For Peer Review 21 22 23 24 Purification 25 26 27 28 29 Refolding 30 31 32 33 34 35 Further purification 36 37 38 Figure 1: Flow diagram for the protein recovery expressed as inclusion bodies in E. coli. – IB: Inclusion bodies. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 k k2 2 1 U I N 3 k4 4 k 5 3 6 7 A 8 9 Figure 2: Simplified refolding pathway of a denatured-reduced protein. – U: unfolded and reduced protein, I: 10 intermediate state, N: native state, A: aggregated state, k: reaction rate constant. 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 1,0 3 α−α−α− lactalbumin 4 scFv 5 0,8 6 7 0,6 8 9 10 0,4 11 12 13 0,2 14 yield refolding Relative 15 16 0,0 17 0,0 0,2 0,4 0,6 0,8 1,0 1,2 18 19 Initial concentration of denatured protein (mg/ml) 20 For Peer Review 21 Figure 3: Relative refolding yield dependent on the initial concentration of denatured protein for α-lactalbumin 22 and a single-chain antibody fragment (scFv). 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 Static mixer Plug flow reactor 2 3 4 Solubilized inclusion 5 bodies 6 7 8 9 Refolding 10 tank 11 12 13 14 15 16 17 18 19 Recirculation stream 20 For Peer(refolding Review buffer) 21 22 Figure 4: Schematic illustration of a refolding device including a static mixer and plug flow reactor [39]. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 1 2 3 4 4 F 5 c0´ 7 6 7 c1´ 12 8 c0, F 0 * 9 c1 (c1 ), F 1 c5´, F 8 10 c0, v 0 c4´ c4, F 6 11 12 11 13 14 5 15 16 17 c4, F 4 18 10 19 Soluble 20 For Peer Reviewaggregates 21 c , F 22 2 2 c3, F 3 23 c4, F 5 Monomer Regenerate 24 25 7 6 26 9 27 8 28 29 30 Figure 5: Experimental setup of continuous refolding by annular chromatography with recycling of aggregates. 31 1 feed pump delivering denatured protein; 2 mixer for blending of fresh feed with recycled feed after 32 concentration by tangential flow filtration; 3 reaction loop; 4 eluent pump for the annular chromatography 33 system; 5 annular chromatography system (a) 6 collecting device for regenerate fractions; 7 collecting device for 34 monomeric protein fraction; 8 tangential flow filtration device; 9 permeate outlet; 10 vessel for collection of 35 concentrated aggregates and 11 recycling pump; 12 pump for delivering regeneration buffer. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 Table 1: Overview of additives for refolding of proteins, patents are bold 2 3 Additive Concentration Range Reference 4 L-Arginine 0.4 – 0.8 M 52,53,54 5 Urea ≤ 2.0 M 51 6 7 Guanidine hydrochloride ≤ 1.0 M 51 8 L-Argininamide ≤ 2.0 M 59 ,60 9 Ethylammonium nitrate 0.5 – 50.0 % 61 ,62 10 (EAN) 11 Secondary and tertiary amines ≤ 1.5 M 63 12 13 Fluorine derivatives 0.5 - 95 % 64 14 Cyclodextrin ( α-,β-,γ-) 5.0 - 10.0 % 66 ,67 15 Ionic liquid 0.25 – 5.0 M 75 16 Cycloamylose *) 16 mM 69 70 17 Alginate*) 0.25-3.0 % 71, 72 18 Reversed Micelles (AOT)*) 73 19 20 *) stripping agent whenFor a surfactant Peer is used for stabilisationReview of protein 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 Table 2: Overview of patents and literature for various refolding techniques. 5 6 7 Homogenous and heterologous techniques Patents Literature reference 8 9 Solubilization of inclusion bodies 44,45,49,50 47,48 10 Batch dilution 32,33 11 12 Continuous dilution 28,29,34 30,31,32,33,41 13 For Peer Review 14 Additives 59, 61,63,64,65,66,70 51,52,53,60,62,67,68,69,71,72,73,74 15 16 Oscillator and other mixing devices 36,38,39 37 17 On column refolding 92,93,105 91,94,96, 99,103 18 19 Immobilized folding catalysts 108* 104,109,110 20 21 High pressure refolding 82,84,86,87 52,81,85 22 23 *Application has been withdrawn 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 22 45 Wiley-VCH 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 23 of 28 Biotechnology Journal

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